This invention relates to a method of improving the production of biomass or a desired product from a cell by inducing conversion of ATP to ADP without primary effects on other cellular metabolites or functions. The invention also relates to a method of optimizing the production of biomass or a desired product from a cell utilizing this first method. The desired product may for example be lactic acid produced by lactic acid bacteria and ethanol or carbon dioxide produced by yeast.
A wide range of microorganisms are used for the production of various organic compounds and heterologous proteins. One example hereof is the production of lactic acid and other organic compounds by the lactic acid group of bacteria, which results in the acidification and flavouring of dairy products, better known as cheese and yoghurt production.
From the microorganism""s point of view, the organic compounds which are excreted from the cells are often merely the by-product of a process that is vital to the cells: the production of various forms of free energy (ATP, NAD(P)H, membrane potential, etc.). Therefore, although many of the microorganisms which are being employed in these processes are reasonably well suited for the purpose, there is still a great potential for optimizing the productivity of these organisms when looking from the bioreactor point of view. Likewise, the production of heterologous proteins by a microorganism is not what the organism was adapted for and also here there is a potential for optimization.
Often when microorganisms are engineered for the purpose of optimizing an industrial production process, the reactions leading to the desired product will affect the delicate balance of co-factors involved in the energy metabolism of the cell. For instance if the glycolytic reactions producing lactate from sugar were somehow to be enhanced (e.g. by overexpressing the glycolytic enzymes) this would automatically lead to the convertion of ADP to ATP. The ratio between the concentrations of ATP and ADP is usually quite high in the growing cell ([ATP]/[ADP] greater than 10), and when the ratio [ATP]/[ADP] changes, the sum of [ATP] and [ADP] still remains virtually constant. Therefore, if in the example above, the enhanced production of ATP changes the [ATP]/[ADP] ratio from 10 to say 30, this will only marginally affect the concentration of ATP. The ADP concentration however will change by a factor of three. The cells will then hardly feel the surplus of ATP but the ADP pool in the cells may be depleted to such an extent that reactions in which ADP is a co-factor or allosteric regulator will be suppressed by the lack of ADP. The result may be that the total flux through the pathway (here through glycolysis) is only marginally increased. In the future, this situation is likely to occur more frequently, as the productivity of bioreactors are optimized by other means, and in these cases, it will be even more important (compared to the normal cell) to regenerate the ADP from ATP, in order to further increase the productivity.
Previously, attempts have been made to decrease the intracellular ATP concentration in yeast, employing sets of reactions which together form futile cycles, see EP patent No. 245 481. Often, the first reaction of a futile cycle is part of the regular metabolic network of the cell, for instance the phosphorylation of a glycolytic intermediate, coupled to the utilisation of ATP. The second reaction, which may also sometimes be part of the metabolic network, then de-phosphorylates the glycolytic intermediate without regenerating the ATP that was consumed in the first process, the overall effect being that a high energy phosphate bond is consumed. The limited success that this strategy has had so far, is probably due to the fact that it is impossible to obtain a significant futile flux without decreasing the concentration of the phosphorylated intermediate, thereby disturbing the cellular function and ultimately the growth. In addition, when the approach is to decrease the concentration of a glycolytic intermediate, this will effectively remove the substrate for the remaining part of the glycolysis, which will often result in a decreased flux through this pathway, rather than the desired increased flux.
Other strategies have been to use chemicals such as dinitrophosphate to stimulate the activity of the plasma membrane H+-ATPase by the addition of uncouplers of the membrane potential, or to genetically express the enzyme acid phosphatase in the cytoplasm, an enzyme that will remove phosphate groups from organic metabolites and proteins. However, both of these approaches suffer from the same inherent problem: they are unspecific and a range of cellular reactions/concentrations may be affected. For instance, the acid phosphatase will remove phosphate groups from essential metabolites and proteins, thus disturbing various metabolic fluxes and metabolic regulation. The uncoupling of the plasma membrane H+-ATPase will disturb the intracellular pH in addition to the gradient of numerous ions across the cytoplasmic membrane. Besides, the addition of chemicals such as dinitrophosphate is undesirable for most purposes.
The idea of the invention is to use a highly specific and clean way to increase the intracellular level of ADP, which does not suffer from the limitations described above: to express in a well-controlled manner an enzyme that has ATP-hydrolytic activity in the living cell without producing other products and without coupling this activity to energy conservation. Such an enzymatic activity is of course not likely to be found in a normal cell, because the cell would then loose some of its vital energy reservoir.
Accordingly the present invention provides a method of improving the production of biomass or a desired product from a cell, the method being characterized by expressing an uncoupled ATPase activity in said cell to induce conversion of ATP to ADP without primary effects on other cellular metabolites or functions, and incubating the cell with a suitable substrate to produce said biomass or product.
One of the normal enzymes that comes closest to the ideal ATP-hydrolyzing enzyme, is the membrane bound H+-ATPase. This huge enzyme complex consists of two parts, the membrane integral part (F0) and the cytoplasmic part (F1). Together the two parts couples the hydrolysis of ATP to ADP and inorganic phosphate (Pi), to translocation of protons accross the cytoplasmic membrane, or vice versa, using the proton gradient to drive ATP synthesis from ADP and Pi.
The method of the invention is conveniently carried out by expressing in said cell the soluble part (F1) of the membrane bound (F0F1 type) H+-ATPase or a portion of the F1 exhibiting ATPase activity.
The membrane bound H+-ATPase complex is found in similar form in prokaryotic as well as eukaryotic organisms, and thus F1 and portions thereof expressing ATPase activity can be expressed in both prokaryotic and eukaryotic cells.
The organism from which the F1 ATPase or portions thereof is derived, or in which the F1 ATPase or portions thereof is expressed, may be selected from prokaryotes and eukaryotes, in particular from bacteria and eukaryotic microorganisms such as yeasts, other fungi and cell lines of higher organisms, in particular baker""s and brewer""s yeast.
A particularly interesting group of prokaryotes to which the method according to the invention can be implemented, i.a. in the dairy industry, are lactic acid bacteria of the genera Lactococcus, Streptococcus, Enterococcus, Lactobacillus and Leuconostoc, in particular strains of the species Lactococcus lactis and Streptococcus thermophilus. Other interesting prokaryotes are bacteria belonging to the genera Escherichia, Zymomonas, Bacillus and Pseudomonas, in particular the species Escherichia coli, Zymomonas mobilis, Bacillus subtilis and Pseudomonas putida. 
In an expedient manner of carrying out the method according to the invention the cell is transformed or transfected with an expression vector including DNA encoding F1 or a portion thereof exhibiting ATPase activity under the control of a promoter functioning in said cell, and said DNA is expressed in the cell. Said DNA encoding F1 or a portion thereof may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said cell.
The F1 part of the bacterial H+-ATPase complex consists of several subunits that together are responsible for catalyzing ATP hydrolysis: the xcex2-subunit is thought to carry the actual hydrolytic site for ATP hydrolysis, but in vitro ATPase activity requires that the xcex2-subunit forms a complex together with the xcex1- and xcex3-subunit (xcex13xcex3xcex23). The activity of this complex is modulated by the xcex5-subunit, so that the in vitro activity of the xcex13xcex3xcex23xcex5 complex is five fold less than the xcex13xcex3xcex23 complex.
In a specific embodiment of the method according to the invention said DNA encoding F1 or a catalytically active portion thereof, is derived from Escherichia coli, Streptococcus thermophilus or Lactococcus lactis and is selected from the group consisting of the gene encoding the F1 subunit xcex2 or a catalytically active portion thereof and various combinations of said gene or portion with the genes encoding the F1 subunits xcex4, xcex1, xcex3 and xcex5 or catalytically active portions thereof.
In particular said DNA encoding F1 or a portion thereof may be selected from the group consisting of the Escherichia coli, Streptococcus thermophilus and Lactococcus lactis genes atpHAGDC (coding for subunits xcex4, xcex1, xcex3, xcex2, xcex5), atpAGDC (coding for subunits xcex1, xcex3, xcex2, xcex5), atpAGD (coding for subunits xcex1, xcex3, xcex2), atpDC (coding for subunits xcex2, xcex5) and atpD (coding for subunit xcex2 alone).
Particularly interesting eukaryotes are the yeasts Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei, and the DNA encoding F1 or a portion thereof may be derived from such organisms and is selected from the group consisting of the gene encoding the F1 subunit xcex2 or a portion thereof and various combinations of said gene or portion with the genes encoding the other F1 subunits or portions thereof.
Vectors including DNA encoding the soluble part (F1) of the membrane bound (F0F1 type) H+-ATPase or a portion of F1 exhibiting ATPase activity, derived from the lactic acid bacteria Lactococcus lactis and Streptococcus thermophilus and from the yeasts Saccharomyces cerevisiae, Phaffia rhodozyma or Trichoderma reesei are also comprised by the invention as well as expression vectors including such DNA under the control of a promoter capable of directing the expression of said DNA in a prokaryotic or eukaryotic cell.
Specific vectors according to the invention are plasmids including DNA encoding the soluble part (F1) of the membrane bound (F0F1 type) H+-ATPase or a portion of F1 exhibiting ATPase activity, said DNA being derived from Lactococcus lactis subsp. cremoris (SEQ ID No. 1), Lactococcus lactis subsp. lactis (SEQ ID No. 6), Streptococcus thermophilus (SEQ ID No. 10), Phaffia rhodozyma (SEQ ID No. 14), and Trichoderma reesei (SEQ ID No. 16).
Further, the invention provides a method of optimizing the formation of biomass or a desired product by a cell, the method being characterized by expressing different levels of uncoupled ATPase activity in the cell, incubating the cell on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product at each level of ATPase expression, and choosing a level of ATPase expression at which the conversion rate is optimized.
Often, but not always, the optimization of a given product flux produced by a cell will entail the attainment of either maximum or minimum conversion rate of a substrate.
In an expedient manner of practicing this method of the invention a number of specimens of said cell are transformed or transfected with their respective expression vector each including DNA encoding a different portion of the cytoplasmic part (F1) of the membrane bound (F0F1 type) H+-ATPase up to and including the entire F1, each portion exhibiting ATPase activity, said DNA in each expression vector being under the control of a promoter functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product in each specimen, and choosing a specimen yielding an optimal conversion rate. In a particular embodiment of this manner, which is especially suited for scientific studies, the promoter in each expression vector is an inducible promoter, and each cell specimen is grown at different concentrations of inducer in order to fine-tune the optimal conversion rate.
In a preferred manner of practicing the above method of optimizing the performance of a cell a number of specimens of said cell are transformed or transfected with their respective expression vector including DNA encoding a portion of the cytoplasmic part (F1) of the membrane bound (F0F1 type) H+-ATPase up to and including the entire F1, said portion exhibiting ATPase activity, said DNA in the respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell, incubating each cell specimen on a suitable substrate, measuring the conversion rate of substrate into biomass or the desired product by each specimen, and choosing a specimen yielding an optimal conversion rate. In a more preferred embodiment of this manner, which is well suited to establish an optimal production strain, the respective expression vectors include DNA encoding different such portions of F1 up to and including the entire F1, each DNA in respective expression vectors being under the control of each of a series of promoters covering a broad range of promoter activities and functioning in said cell.
Also in this method of the invention the DNA encoding a portion of F1 up to and including the entire F1 may be derived from a prokaryotic or a eukaryotic organism, and it may be either homologous or heterologous to said organism. The specific DNAs mentioned above may also conveniently be employed in this method.